Transcription factors in muscle atrophy caused by blocked neuromuscular transmission and muscle unloading in rats

Abstract

The muscle wasting associated with long-term intensive care unit (ICU) treatment has a negative effect on muscle function resulting in prolonged periods of rehabilitation and a decreased quality of life. To identify mechanisms behind this form of muscle wasting, we have used a rat model designed to mimic the conditions in an ICU. Rats were pharmacologically paralyzed with a postsynaptic blocker of neuromuscular transmission, and mechanically ventilated for one to two weeks, thereby unloading the limb muscles. Transcription factors were analyzed for cellular localization and nuclear concentration in the fast-twitch muscle extensor digitorum longus (EDL) and in the slow-twitch soleus. Significant muscle wasting and upregulation of mRNA for the ubiquitin ligases MAFbx and MuRF1 followed the treatment. The IkappaB family-member Bcl-3 displayed a concomitant decrease in concentration, suggesting altered kappaB controlled gene expression, although NFkappaB p65 was not significantly affected. The nuclear levels of the glucocorticoid receptor (GR) and the thyroid receptor alpha1 (TRalpha1) were altered and also suggested as potential mediators of the MAFbx- and MuRF1-induction in the absence of induced Foxo1. We believe that this model, and the strategy of quantifying nuclear proteins, will provide a valuable tool for further, more detailed, analyses of the muscle wasting occurring in patients kept on a mechanical ventilator.

Figure 1

Transcription factors quantified in nuclear extracts using ELISA. The bars represent the relative change for NMB as a percentage of control in A. EDL and B. Soleus. *P < 0.05, ***P < 0.001. Error bars represent SEM.

Figure 2

Cross sections of EDL muscle from an NMB rat showing Myf-5 (green), MyHC type I (red) and nuclei (blue). Myf-5 shows a clear nuclear localization and a predominant staining in the cytoplasm of type II fibers. (A) Myf-5 staining only, (B) composite view of all fluorophores, scale bar 50 μm. A higher magnification of the area in (A) and (B) is shown in (C) and (D). Some nuclei are intensely stained for Myf-5 (arrows) while it is absent in others (arrowhead), scale bar 20 μm.

Figure 3

Cross sections of soleus muscle. (A), (E), (I), and (M) show the localization of glucocorticoid receptor, NFκB MEF-2, and Myogenin, respectively. In (B), (F), (J), and (N) the nuclei are displayed together with respective transcription factors. In (C), (G), (K), and (O), corresponding images display the type I fibers in red and type II fibers unstained (some marked by arrowheads). The NFκB concentration appears with different intensity among the type II fibers (areas of less intensity are indicated by arrows). The scale bar for A–L is 100 μm and the scale bar for M–P is 50 μm.

Figure 4

(A) Localization of NFATc1 (green) in a longitudinal section of EDL muscle from a control rat. A faint staining of nuclei and cytoplasm is visible in the muscle fibers. Note also the striated pattern suggesting an association of NFATc1 to sarcomeric proteins. The most intense staining is found in the vascular smooth muscle cells. (B–D) Cross section of EDL muscle from a control rat. (B) Localization of NFATc1 is shown in green and nuclei in blue; (C) distribution of type I fibers (red); (D) composite image. Scale bar for A is 20 μm and for B–D 50 μm.

Figure 5

mRNA levels of MuRF1 (A) and MAFbx (B) in EDL and soleus muscle tissues. NMB, n = 4; controls, n = 8. *P < 0.05, **P < 0.01, ***P < 0.001.